Levels of Service

The Client will supply raw tissue/cells/media with a request for sample processing with NMR and/or MS analyses

OR the Clients supply metabolite extracts with a request for standard NMR and/or MS analyses.

Then the Center prepares the extracts accordingly, records the appropriate spectra under standard operating conditions, and returns the raw data to the Client

OR the Center prepares the extracts accordingly, records the appropriate spectra under standard operating conditions, and reduces the data to a list of quantified identified metabolites with respect to the current databases, with isotopomer and isotopologue distributions when appropriate.

Experimental Design

The Client consults with the Center leadership on the overall goals to develop a detailed plan for the experimental design, numbers of samples, sample handling and integrity, numbers of analytes and analytical platforms, number and types of tracers to be used.

Biostatistical Analysis

The Center will assist in various biostatistical analyses including metabolic profiling and sample classification.

Guidance on Mechanism-Based Analysis and Omics Integration

The Center will assist in mechanism-based biochemical interpretation, which includes relevant metabolic pathway analysis and integration of metabolic data with other streams of information such as transcriptomics, proteomics, and functional annotations.

Beyond rudimentary guidance, this will require collaboration.

Custom Services

The Center will work with Clients to develop new methods and standard operating procedures that address their specific needs.

In vivo NMR experiments (e.g. preclinical mouse model studies) will always be under this category.

Custom Services will usually incur the highest rates, and will require collaboration where new methods or assays are developed

Academic Rate Schedule

All rates stated are based on estimates from recharge rate calculation spreadsheets (RRC) that annually balance Center expenditures with projected income for each chargeable operation (e.g. each instrument, sample preparation technique, informatics computer usage).

Targeted analysis of a defined, short list of analytes, e.g. 10 specific isobaric lipids. Usually performed in a fashion that may preclude data mining for non-target analytes. [Note: Proximate type of analysis, such as total diacyl glycerides, is NOT this rate, but is rather Tier B (Rate 2).]

Targeted analysis of a defined, short list of analytes, e.g. 10 specific nucleotides. Usually performed in a fashion that may preclude data mining for non-target analytes. [Note: Proximate type of analysis, such as total purine nucleotides, is NOT this rate, but is rather Tier E (Rate 5).]

$45.00

7

Tier G FTMS

Multiply labeled tracer analysis (for example 15N13C Gln)

Analysis is complicated and time consuming as more than one stable isotope enrichment is investigated. Likely to incur Data Processing (Rate 31) surcharges.

For many types of samples, it is best to normalize analyte results to total protein.

$20.00

28

Robotic Liquid Handler

Robotic Liquid Handler

Standalone use of the Tecan Fluent 780 liquid handler

$40.00

29

Particle Analyzer

Nanosight Particle Counter/Sizer

Counts particles with size distribution, e.g. microvesicles.

$10.00

30

Tubewriter

Tubewriter labeler

Automated labware labeler capable of 1 & 2D barcodes.

$10.00

31

Data Processing

Data Processing

Data processing where not included above, or as a surcharge.

$40.00

32

Ancillary Labor

Ancillary Labor

Method development in excess of rates 1-31, extensive advising and instruction

$60.00

33

Ancillary Supplies

Ancillary Supplies

Method development in excess of rates 1-31, extensive advising and instruction

at cost

34

Bench Fee

Half Bench for sample processing, 11 linear ft per day

For clients lacking sufficient facilities may utilize CESB wet bench. User must meet all regulations and requirements for lab bench work at UKY, including any specific requirements for working in CESB space (which handles e.g. human subject samples).

$66.00

**available only to academic users, not to fee for service commercial users

The Seahorse XRF provides information about the rate of proton extrusion (ECAR) and the rate of oxygen consumption (OCR) by adherent cells under normoxic conditions.

By varying the extracellular nutrient source (e.g. glucose, glutamine, fatty acids) and with the use of specific inhibitors, it is possible to estimate net lactic fermentative flux, glycolytic reserve, respiration potential and the degree of coupling to oxidative phosphorylation. Such information is extremely valuable in designing metabolomics analyses or whether metabolism is a significant correlate of the biological function of interest.

The consultation will then focus on the experimental design, which will also need to include biostatisticians for estimating sample sizes.

It is also important to establish whether the user will do their own sample work up, what analytical techniques are needed, and what level of data reduction is required.

The Center has SOPs. Questions that cannot be answered using these SOPs will be considered in terms of development time to establish specific metabolic assays.

This is relevant mainly in the context of a small number of metabolites (targeted metabolomics).

Costs.

What are the costs associated with the samples being analyzed?

What are the costs of the metabolomics experiments, including tracers?

It is VERY common for data analysis to take 90% of the metabolomics labor, so the researcher should be prepared to discuss the time frame and cost structure with this consideration, versus obtaining data analysis training for their own laboratory.

We have found, however, that the latter approach still incurs considerable labor for the CESB/RCSIRM staff, as data analysis is highly nuanced and extensive consulting is still typically needed.

Once an experimental approach has been worked out, the user will be introduced to laboratory personnel who will carry out the analyses and oversee the project progress- this person will then be the primary contact.

Data collection is not the rate limiting step. Global analyses often produce hundreds to thousands of identifiable and quantifiable compounds or features, but this means the reduction of raw data is extremely time consuming.

Useful Publications

Examples of the use of stable isotope resolved metabolomics can be found in the publications listed below:

An example study and pipeline

What is the role of tumorin in tumor development and survival in TNBC?

The goal is to understand how central metabolism is impacted by the knockdown, including sources of NADPH needed for proliferation. The user may already have information from Seahorse analysis, and phenotypic effects of the knockdown. Cell cycle distribution analysis is also important for the overall interpretation.

The results can be interpreted in terms of specific networks related to cell growth or survival, with limited flux information (exclusively in this design for inputs and outputs).

Data Processing and Analysis

The data comprise several components, as follows.

Metadata that describe in exact detail the entire workflow from sample receipt to final products. No useful results can be obtained without these data. An Excel spreadsheet is required for these data, and a template is provided.

Raw analytical data, i.e. the streams of bits coming from the instruments. For FT-MS and NMR these represent digitized electrical signals in the form of free induction decays comprising both real and imaginary parts. For other MS data, these are digital representations of ion counts.

Raw analytical data have to be transformed into a usable form, which for FT-MS and NMR is the discrete fourier transform and associated digital processing to suppress truncation artefacts, optimize signal-to-noise ratios etc. The resulting output is a spectrum of intensity versus frequency. For NMR, the frequency is usually transformed to chemical shift, in ppm, that is independent of magnetic field strength. For FT-MS, the frequencies are mapped onto an m/z range.

Intensities (ordinate values) must be internally normalized to obtain amounts of materials (i.e. numbers of moles of substances or of ions), and back calculated to the values associated with the original spectrum, on an agreed upon measure of the amount of that species (such as biomass weight, protein mass etc.). This absolutely requires accurate metadata. The amounts are proportional to peak areas (or volumes) NOT peak heights; appropriate numerical or analytical integration procedures must be correctly applied, taking due account of baseline drift, phasing errors and peak overlap.

For isotopomer and isotopologue analyses, the intensities are usually expressed as mole fractions (enrichments). As these are ratios, normalization to cell amount is not needed. For MS, the natural abundance needs to be corrected. Software is available for this [cf. Moseley (2010) Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinformatics 11,139]

Spectral features need to be mapped onto identifiable molecules (assignment), using the available spectral information, and by reference to our databases.

For profiling typically one is concerned with case-control comparisons, which require large numbers of specimens (each unique). Multivariate statistics are generally appropriate for initial analyses- are the groups different? What is different about these groups? PCA and OPLSDA (SimcaP) may be used.

Normalization. To compare case and control, the quantity of each metabolite must be normalized to the appropriate amount of specimen. Cell number is generally not appropriate as cell volumes vary widely among types, and also in response to treatment. Total biomass or a surrogate is appropriate (e.g. dry weight, total protein).

Total DNA may not be appropriate in a case-control study because the amount of DNA per cell varies twofold during the cell cycle, and the control and treated samples do not necessary have the same cell cycle distribution. Comparison of different cell types is then further compromised where there are different numbers of chromosomes present (diploid G1 normal cell, triploid cancer cell, tetraploid cardiomyocytes arrested at G2/M).

With SIRM studies, a question is often what pathways were impacted, which requires pathway tracing (SIRM) and biochemical expertise.

Quantitative analyses may also be carried out (e.g. what is the rate of nutrient utilization and waste product excretion). Kinetic models (flux analysis) based on enzymology can also be applied where needed. These studies need careful consideration of the time dependence of the biomass as a function of time for accurate normalization of rates. Flux: the number of moles of nutrients (e.g. glucose, glutamine) consumed and the number of moles of product excreted (e.g. lactate, alanine, glutamate) is measured as a function of time, producing a time course of consumption and excretion. To determine rates, it is essential to normalize to the functional unit of metabolism which is the amount of enzymes present in the system. This is proportional to the concentration of the enzymes times the relevant intracellular volume (unknown).

With tracers, the time course of the isotopomer distributions can be determined, as can the fraction of glucose (glutamine) consumed that is converted to excreted product (e.g. lactate, alanine, glutamate).

Further statistical analyses if needed should be carried out by statisticians versed in multivariate analyses.